Nucleic acid amplification device, nucleic acid amplification method, and chip for nucleic acid amplification

ABSTRACT

The present invention provides a reciprocal-flow-type nucleic acid amplification device comprising: 
     heaters capable of forming a denaturation temperature zone and an extension/annealing temperature zone;
 
a fluorescence detector capable of detecting movement of a sample solution between the two temperature zones;
 
a pair of liquid delivery mechanisms that allow the sample solution to move between the two temperature zones and that are configured to be open to atmospheric pressure when liquid delivery stops; a substrate on which the chip for nucleic acid amplification according to claim  2  can be placed; and a control mechanism that controls driving of each liquid delivery mechanism by receiving an electrical signal from the fluorescence detector relating to movement of the sample solution from the control mechanism; the device being capable of performing real-time PCR by measuring fluorescence intensity for each thermal cycle.

TECHNICAL FIELD

The present invention relates to a nucleic acid amplification device, anucleic acid amplification method, and a chip for nucleic acidamplification.

BACKGROUND ART

The detection of nucleic acids is central in various fields, such asresearch and development of medicine, forensic science, clinical tests,and identification of types of agricultural products and pathogenicmicroorganisms. The capability to detect various diseases, such ascancer, infection of microorganisms, gene markers, etc., throughmolecular phylogenetic analysis is a universal technique for thediagnosis of diseases and the risk of developing diseases, the searchfor markers, the evaluation of food and environmental safety, the proofof crimes, and many other techniques.

One of the most powerful basic technologies for detecting a small amountof nucleic acid, which is a gene, is a method for analyzing a productobtained by exponentially replicating a part or the whole of a nucleicacid sequence.

PCR is a potent technique for selectively amplifying a specific regionof DNA. With PCR, one can quickly produce millions of copies of thetarget DNA sequence in a template DNA from a single template DNAmolecule. In PCR, a two- or three-phase temperature cycle, called a“thermal cycle,” is repeated to sequentially perform the followingindividual reactions: denaturation of DNA into single strands; annealingof primers to the denatured DNA single strands; and extension of theprimers by a thermostable DNA polymerase enzyme. This cycle is repeateduntil enough copies for analysis have been obtained. In principle, eachcycle of PCR can double the number of copies. In reality, as the thermalcycle continues, the concentrations of required reactants are reduced,so that the buildup of amplified DNA products eventually ceases. Forgeneral details concerning PCR, see “Clinical Applications of PCR,”(edited by) Dennis Lo, Humana Press (located in Totowa, N.J., 1998), and“PCR Protocols—A Guide to Methods and Applications,” (edited by) M. A.Innis et al., Academic Press Inc. (located in San Diego, Calif.)(1990).

Although PCR is a potent technique for selectively amplifying thedesired DNA, confirmation by gel electrophoresis, etc., is necessary toconfirm the amplified DNA after the completion of PCR. Therefore, as animproved PCR method, real-time PCR evolving or quenching fluorescence inaccordance with the amplified amount of the desired DNA was developedand the presence or absence of the desired DNA in a sample became easilyconfirmed. In conventional PCR methods, when the amount of template DNAin a sample before PCR exceeds a certain amount, the amount of amplifiedDNA after PCR often plateaus and the template DNA amount before PCRcannot be quantified. However, in the real-time PCR method, before PCRplateaus, the amount of amplified DNA during PCR can be detected inreal-time; therefore, the amount of template DNA before PCR can bequantified from the DNA amplification state. Accordingly, the real-timePCR method is also called a quantitative PCR method.

Quantification of the target DNA amount by real-time PCR has particularclinical utility and is used, for example, for monitoring changes inviral load to confirm therapeutic effects against virus infection, suchas the AIDS virus (HIV). DNA quantification by real-time PCR is alsoeffective for the diagnosis of opportunistic infections, such asherpesvirus (HHV), with which many subjects have been subclinicallyinfected since infancy and which develop as a result of weakenedphysical strength or the like.

PCR and real-time PCR are potent methods for exponentially amplifyinggenes by thermal cycling. General-purpose thermal cycle devices used inPCR are slow in temperature control due to the huge thermal capacity ofthe aluminum block used as a heater, and it conventionally takes 1 to 2hours, or even more in some cases, to perform 30 to 40 cycles of PCR.Accordingly, even with the use of a state-of-the-art genetic testingdevice, analysis usually takes a total of 1 hour or more. Increasing thespeed of PCR has been a great challenge since the development of thistechnique. Various methods have been developed for increasing the speed.Methods for increasing the speed of thermal cycling samples areclassified into the following three types of methods.

In the first method, a sample solution is introduced into the device andtemperature cycling is performed with time while the solution is held inthe same portion (Non-patent Literature (NPL) 1 and Patent Literature(PTL) 1). This method is intended to increase the speed of thermalcycling by reducing the sample amount and thereby reducing the thermalcapacity. However, there is a limit to the reduction of thermal capacityof the chamber or heater itself. Therefore, it takes at least about 30seconds per cycle to perform sufficient amplification reactions. Ittakes 15 minutes or more to complete the PCR reaction even with thefastest device.

The second method is called continuous-flow PCR. In this method, asample solution passes through a microchannel to move through multipletemperature zones spaced apart from each other while the solution iscontinuously fed without stopping. Among such continuous-flow PCRmethods, a system of quickly controlling the sample temperature bypassing a sample solution through serpentine microchannels above threeheaters controlled at constant temperatures is known (NPL 2). Since thiscontinuous flow PCR method does not require temperature changes ofexternal devices, such as containers and heaters, the fastesttemperature control can be theoretically expected. In an extremely fastcase, amplification of DNA is effectuated in about 7 minutes. However,to perform quantitative real-time PCR by using continuous-flow PCR, amechanism that enables fluorescence observation of the whole region ofthe serpentine microchannels or 30 to 50 regions of each serpentinemicrochannel in the same temperature zone is necessary. Specifically, anexcitation light source that can uniformly irradiate a wide region and ahigh-sensitivity video camera or a line scanner for fluorescenceobservation is necessary, which unavoidably results in a large andexpensive system structure.

In the third method, like the second method, multiple temperature zonesspaced apart from each other are connected by microchannel(s), and asample solution is reciprocally moved through the same microchannel(s)in such a manner that the sample solution is stopped in each temperaturezone for a certain period of time, and heated (Patent Literature (PTL)2). This method is advantageous in that the time of contacting a samplewith each temperature zone can be freely set to perform thermal cycling.However, in order to introduce a sample and pump it to these temperaturezones reciprocally or rotationally, many integrated valves and pumps, aswell as detectors for observing the position of the sample solution, arenecessary to inhibit the sample solution from unwillingly moving from adesirable temperature zone position in the microchannel due to theexpansion of tiny air bubbles generated in the sample heated at a hightemperature side and/or a difference in vapor pressure generated on thegas-liquid interface when the sample solution moves through amicrochannel having a temperature gradient of from about 95° C. or morefor a denaturation reaction to about 60° C. for an annealing reaction,and device miniaturization was difficult (NPL 3 and 4 and PTL 3).

The market for genetic testing using PCR/real-time PCR devices isfavorably growing. In particular, genetic testing for infectiousdiseases, such as viral hepatitis, sexually transmitted diseases, andinfluenza, is also rapidly spreading in Japan. The usefulness of genetictesting for cancer treatment has become apparent. For example, EGFR genemutation can be used as a rule of thumb for applying the cancer agentIressa. Therefore, genetic testing for the EGFR gene, K-ras gene,EWS-Flil gene, TLS-CHOP gene, SVT-SSX gene, and c-kit gene in lungcancer, pancreatic cancer, etc., has recently been covered by insurance.

In PCR, primers are attached to a template DNA and the target DNAlocated between the primer sequences is specifically detected by a DNApolymerase. Although PCR can be used for detecting DNA, PCR cannotdirectly detect RNA. Accordingly, in order to detect RNA viruses, suchas influenza viruses or noroviruses, PCR is performed aftercomplementary cDNA is synthesized by reverse transcriptase using RNA asa template; that is, so-called RT-PCR is performed. Thus, substantiallya two-stage step must be performed. Furthermore, PCR and RT-PCR need arapid temperature rise and fall and thus require a special incubator.Therefore, there is a problem in that the adaptation of PCR and RT-PCRto automation is not easy.

In recent years, multiplex PCR, which simultaneously amplifies multiplegenetic regions by using multiple pairs of primers in one PCR system,has been attracting attention. Real-time multiplex PCR, which wasdeveloped from a multiplex PCR, is intended to individually detect andquantify multiple different target genes with less influence (crosstalk)of other target genes and without compromising sensitivity. However, ithas been reported that two or more quantitative multiplex reactions areoften difficult due to the problem of overlapping wavelengths and kindsof labelable fluorescent substances.

At present, genetic testing is taken to and performed at laboratories oranalysis centers. However, if a high-speed, real-time PCR device thatcan quickly perform genetic testing on the spot is available, the courseof treatment and countermeasure can be determined on the spot.Therefore, such a device is considered to be an epoch-making techniquethat can replace current genetic testing equipment. In particular, as aquarantine measure to prevent pandemics of foot-and-mouth disease,highly pathogenic influenza, and the like, quick and correct judgment onthe spot and prevention of spreading of secondary infection areimportant. The need for such a high-speed, real-time PCR device istremendous.

In particular, to realize a service that allows genetic testing to beimmediately performed in a clinical setting or on the spot where aninfectious disease occurs, a high-speed and highly portable real-timePCR device that can operate at low cost is necessary.

CITATION LIST Patent Literature

-   PTL 1: Canadian Patent Application Publication No. 2479452-   PTL 2: JP2003-200041A-   PTL 3: WO2006/124458

Non-Patent Literature

-   NPL 1: Neuzil et al. (Lab Chip 10:2632-2634 (2010))-   NPL 2: Kopp et al. (Science 280: 1046-1048 (1998))-   NPL 3: Chiou et al. (Anal Chem 73: 2018-2021 (2001))-   NPL 4: Brunklaus et al. (Electrophoresis 33: 3222-3228 (2012))

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a small, nucleic acidamplification device that can be carried for use on the spot and thatcan perform high-speed, real-time PCR, a plate for the device, and anucleic acid amplification method.

Solution to Problem

To achieve an increased reaction efficiency and provide a smalleramplification device, the present invention provides areciprocal-flow-type, high-speed, real-time, nucleic acid amplificationdevice that comprises: two temperature zones disposed on a flat surface;a microchannel contacted to each temperature zone, both ends of themicrochannel being configured to be open to atmospheric pressure whenblowers, fans, etc. are stopped; and liquid delivery mechanisms by whicha plug sample solution is reciprocated between precise positions in thetemperature zones through the microchannel to perform thermal cyclingwhile simultaneously confirming the passing of the PCR solution andmeasuring fluorescence intensity for each thermal cycle. In anotherexemplary embodiment, the nucleic acid amplification method comprisesconverting RNA to cDNA by reverse transcription and subjecting the cDNAto PCR.

Specifically, the present invention provides the following nucleic acidamplification devices, nucleic acid amplification methods, and chips.

(1) A reciprocal-flow-type nucleic acid amplification device comprising:heaters capable of forming a denaturation temperature zone and anextension/annealing temperature zone;a fluorescence detector capable of detecting movement of a samplesolution between the two temperature zones;a pair of liquid delivery mechanisms that allow the sample solution tomove between the two temperature zones and that are configured to beopen to atmospheric pressure when liquid delivery stops;a substrate on which a chip for nucleic acid amplification can beplaced; anda control mechanism that controls driving of each liquid deliverymechanism by receiving an electrical signal relating to movement of thesample solution from the fluorescence detector,the device being capable of performing real-time PCR by measuringfluorescence intensity for each thermal cycle.(2) A chip for nucleic acid amplification comprising at least onemicrochannel, the microchannel comprising:curved-microchannels each for the denaturation temperature zone and theextension/annealing temperature zone of the nucleic acid amplificationdevice according to (1);a linear intermediate-microchannel connecting the curved-microchannels;andconnections at both ends of the microchannel,the connections being connectable to the liquid delivery mechanisms ofthe nucleic acid amplification device according to (1).(3) The nucleic acid amplification device according to (1), wherein theliquid delivery mechanisms are microblowers or fans.(4) A nucleic acid amplification method comprising the following steps:step 1: placing the chip for nucleic acid amplification according to (2)on the substrate according to (1) in such a manner that the denaturationtemperature zone includes one curved-microchannel and theextension/annealing temperature zone includes anothercurved-microchannel;step 2: introducing a sample solution into the microchannel;step 3: connecting a pair of liquid delivery mechanisms to liquiddelivery mechanism connections at both ends of the microchannel; andstep 4: reciprocating the sample solution between the twocurved-microchannels of the microchannel by the liquid deliverymechanisms to perform thermal cycling, and simultaneously measuring thefluorescence intensity of the sample solution and confirming the passingof the sample solution for each thermal cycle using at least onefluorescence detector in the intermediate-microchannel to performreal-time PCR.(5) The nucleic acid amplification method according to (4), wherein themeasurement of fluorescence intensity is performed by simultaneouslymeasuring two or more fluorescent wavelengths to simultaneouslymeasuring real-time PCR of multiple genes in one microchannel.(6) The nucleic acid amplification method according to (4) or (5),wherein the measurement of fluorescence intensity is performed using acalibration curve obtained from the number of cycles Ct derived from afluorescence intensity matrix per thermal cycle (a two-dimensional arrayof an amplification curve).(7) The nucleic acid amplification method according to any one of (4) to(6), wherein the nucleic acid amplification method is selected from thegroup consisting of polymerase chain reaction (PCR), reversetranscription PCR (RT-PCR), multiplex PCR, multiplex RT-PCR, real-timePCR, and real-time RT-PCR.(8) The nucleic acid amplification method according to any one of (4) to(7), comprising performing interruption analysis, which is made possibleby forming two or more microchannels on a flat substrate in such amanner that the operation of liquid delivery through each of themicrochannels can be independently controlled.(9) The nucleic acid amplification method according to any one of (4) to(8), the method comprising:connecting an end of a filtered pipette tip of a micropipette to one ofthe connections so as to introduce a sample solution into themicrochannel;removing the micropipette with the pipette tip being connected to theconnection, and then connecting the pipette tip to one of the liquiddelivery mechanisms.(10) The nucleic acid amplification method according to any one of (4)to (9), wherein the volume of the sample solution introduced into themicrochannel is in the range of 5 μL to 50 μL.(11) The chip for nucleic acid amplification according to (2), the chipbeing for use in the nucleic acid amplification method according to anyone of (4) to (10).

Advantageous Effects of Invention

According to the present invention, since two liquid delivery mechanisms(preferably fans) and one fluorescence detector are provided for eachmicrochannel, a low-cost and compact portable device can be realized.

Furthermore, since the sample solution is reciprocated between twotemperature zones, faster real-time PCR can be achieved bysimultaneously and quickly performing the confirmation of passing of thePCR solution and the measurement of fluorescence intensity for eachthermal cycle.

In conventional methods using thermal cycling by reciprocating a samplesolution, a pressure source, such as a syringe pump, is connected to amicrochannel, and a plug-like sample solution is reciprocated byrepeating pressurization and depressurization. In this process, theinside of the microchannel at the side connected to such a pressuresource must be a closed system so as not to leak pressure from theplug-like sample solution in the microchannel. When the force of theplug-like sample solution applied to the gas-liquid interface, which isgenerated by applying or reducing pressure by means of the pressuresource, exceeds the static friction between the plug-like samplesolution and the microchannel internal wall, liquid delivery begins. Onthe other hand, when pressurization or depressurization is stopped tostop the plug-like sample solution, the pressure of the sample solutionacting on the gas-liquid interface remains inside the closedmicrochannel at the pressure source side, and the solution keeps movingfor a while until the energy is completely consumed by kinetic frictionand then stops. In particular, when the sample solution is heated toabout 95° C. or higher for a denaturation reaction as in PCR, theinfluence of changes in the internal pressure of the sample solution,such as viscosity changes and the formation of tiny air bubbles, isgreat and the amount of movement after stopping the pressure source,such as a pump, varies widely. To stop the sample solution at a preciseposition, several measures, such as dedicated valves for releasing theinternal pressure of the microchannel as well as complicated operationof the pressure source, and dedicated sensors for confirming theposition of the solution, were necessary.

In contrast, although the present invention utilizes applying orreducing pressure in the microchannel by blowing air using blowers,fans, etc., to reciprocate a plug-like sample solution, liquid deliverystops immediately after air blowing using the blowers, fans, etc., isstopped, when or immediately before the solution has reached the preciseposition on each temperature zone. This is because when air blowing isstopped, the internal pressure of the microchannel is instantaneouslyopen to atmospheric pressure and the pressure acting on the plug-likesample solution is lost, whereby liquid delivery stops immediately.Accordingly, even in the absence of multiple valves for releasingpressure for controlling the position of the sample solution, a preciseposition control can be achieved by confirming the passing of the samplesolution only at one point located between each temperature zone.Furthermore, since fluorometry can also be simultaneously performed atthe point where the position of the reciprocated liquid is confirmed foreach cycle, a real-time PCR thermal cycler with the simplest structurewhere only one point in the linear microchannel is used as a detectingpoint can be realized.

In the present invention, a polymerase chain reaction, commonly referredto as PCR, is used. PCR uses multiple cycles of denaturation, annealingof primer pairs to opposite strands, and primer extension toexponentially increase the number of copies of a target nucleic acidsequence. In its variation, called reverse transcription PCR (RT-PCR),reverse transcriptase (RT) is used to make a complementary DNA (cDNA)from mRNA, and the cDNA is then amplified by PCR to produce a largenumber of copies of DNA.

As an RT-PCR reaction, one-step RT-PCR can also be used. One-step RT-PCRis an RT-PCR method capable of performing RT-PCR quickly andconveniently in one step from the incubation in RT to cycling in PCRwithout opening and closing the tube or adding a reagent. In thistechnical field, various kits and protocols for one-step RT-PCR (such asOne Step RT-PCR Mix of QIAGEN) can be used and appropriately selected toperform one-step RT-PCR.

For various other permutations of PCR, see, for example, U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159; Mullis et al, Meth. Enzymol. 155:335 (1987); and Murakawa et al, DNA 7: 287 (1988), each of which isherein incorporated in its entirety by reference.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the configuration of a nucleic acid amplification device.

FIG. 2 shows the configuration of a PCR chip.

FIG. 3 shows amplification of nucleic acids in high-speed, real-timePCR.

FIG. 4 shows a calibration curve of Escherichia coli (E. coli) for usein high-speed, real-time PCR.

FIG. 5 shows retention time plotted against each target DNA length, theretention time allowing sufficient amplification of target DNA withoutexhibiting any changes in Ct values even when annealing time andextension reaction time are shortened.

FIG. 6 shows multiplex PCRs of pathogenic microorganisms A and B and βactin gene.

FIG. 7 shows nucleic acid amplification of RNA comprising a norovirus G1or G2 gene sequence in high-speed, one-step reverse transcriptionreal-time PCR using different reverse transcriptases.

FIG. 8 shows nucleic acid amplification from different initialconcentrations of RNA comprising a norovirus G1 or G2 gene sequence inhigh-speed, one-step reverse transcription real-time PCR.

FIG. 9 shows calibration curves obtained from the number of cycles Ct inwhich fluorescence intensity rapidly amplifies and rises in high-speed,one-step reverse transcription real-time PCR, plotted against RNAcomprising a norovirus G1 or G2 gene sequence.

DESCRIPTION OF EMBODIMENTS

One embodiment of the reactor of the present invention is explainedbelow with reference to FIGS. 1 to 9.

As shown in FIG. 1, the device used in high-speed, real-time PCR isconfigured to comprise a substrate for placing a PCR chip thereon (notshown), a temperature control section for a PCR chip, liquid deliverymicroblowers as liquid delivery mechanisms, a fluorescence detector, acontrol computer as a control mechanism, and a battery for power supply.

The temperature control section for a PCR chip is configured to comprisetwo cartridge heaters disposed in parallel with an interval of 10 mmtherebetween so as to be in contact with the sealing surface side of theserpentine microchannels of a PCR chip without any space therebetween.To control the temperatures of the two heaters, each heater comprises aK-type thermocouple joined thereto.

A cartridge heater 1 is controlled by means of a control computer to atemperature necessary for a DNA denaturation reaction essential for PCR.The temperature (denaturation temperature zone) is preferably 90 to 100°C., and particularly preferably 95° C. A cartridge heater 2 iscontrolled to a temperature necessary for an annealing reaction and anextension reaction of DNA (an extension/annealing temperature zone) by acontrol computer. This temperature is preferably 40 to 75° C., andparticularly 55 to 65° C. The temperature zone for DNA denaturationreaction and the temperature zone for annealing reaction and extensionreaction are preferably controlled at constant temperatures. Forexample, the temperature zones are retained at constant temperatures byPID (proportion-integration-differential) control.

The PCR solution to be delivered is quantified using a micropipette orthe like to the required amount within the range of 5 to 50 μL, and morepreferably 5 to 25 μL. With the PCR sample solution being contained inthe micropipette, the disposable tip of the micropipette is mounted onone end of a microchannel. After the micropipette body is removed, anair pressure tube connected to a liquid delivery microblower isconnected instead and pressure is applied by blowing air, so that asample solution can be fed into the microchannel of the PCR chip.

The PCR sample solution is a premixed product of components necessaryfor PCR with a fluorescent probe, such as TaqMan probe, Cycleave probe,or E Probe®, and a fluorescent dye, such as SYBR GREEN, so as to enablereal-time PCR. As such fluorescent probes, reagent kits for real-timePCR and products synthesized by an outsourcing company can be used.

The fluorescence detector is disposed to measure the fluorescenceintensity at one detecting point on a linear microchannel that isdisposed at the center of each microchannel. When the PCR solutiondelivered from one of the serpentine microchannels by applying pressurepasses through a detecting point, liquid delivery microblowers arestopped, so that the PCR solution can be retained in the otherserpentine microchannel for a certain period of time.

The control computer can be programmed to simultaneously control twomicroblowers connected to each microchannel. While continuouslymonitoring the fluorescence intensity at a detecting point at the centerof each microchannel, the microblowers are alternately switched toalternately move the PCR sample solution to each serpentine microchannelabove each heater for a predetermined period of time and perform thermalcycling. Further, in the real-time PCR method, the control computersimultaneously records changes in fluorescence intensity per cycle,which increases as the target DNA is amplified by thermal cycling in thereal-time PCR method, and calculates the number of cycles (Ct value) inwhich the fluorescence intensity crosses a certain threshold, thusquantifying the initial amount of target DNA.

The PCR chip for use in high-speed, real-time PCR (chip for nucleic acidamplification) is configured to comprise a COP resin substrate, whichcomprises four microchannels formed in parallel by injection molding,and a polyolefin transparent seal applied to the substrate.

Each microchannel is configured to meander and turn back in two sectionswith a width as shown in FIG. 2 and a depth of 700 μm. Each serpentinemicrochannel is turned back four times in such a manner that the linearmicrochannel at the center of each microchannel is sandwiched betweenthe serpentine microchannels, and at least 25 μL of the solution can beaccommodated in each serpentine microchannel portion alone.

The regions encompassed by dotted lines (a denaturation temperature zoneand an extension/annealing temperature zone) in FIG. 2 were heated byheaters for thermal cycling in real-time PCR.

Both ends of the microchannel are individually connected to aperturespenetrating a resin substrate (connections to liquid deliverymechanisms). Even after the entire microchannel-side surface of theresin substrate is joined by a polyolefin transparent seal, the reactionsolution and air can pass through the apertures into each microchannel.

The aperture is configured to permit the mounting of a disposable tipfor micropipettes generally used in biochemistry experiments. After 5 to25 μL of a PCR solution is measured, the disposable tip is directlyconnected, whereby the PCR solution can be introduced without thenecessity of using a special instrument and without contamination, etc.

An interruption analysis is made possible by forming two or moremicrochannels on a flat substrate or disposing in parallel two or moreflat substrates each comprising a microchannel, so that the operation ofliquid delivery through each of the microchannels can be independentlycontrolled.

The microchannel preferably comprises a material that has relativelyhigh thermal conductivity, is stable in the temperature range necessaryfor PCR, is resistant to erosion by electrolytic solutions and organicsolvents, and does not absorb nucleic acids or proteins. Examples ofmaterials include glass, quartz, silicon, and various plastics. Theshape of the microchannel that is in contact with multiple temperaturezones may be not only a linear microchannel but also a curvedmicrochannel, such as a serpentine microchannel having a loop shape, ora spiral microchannel. The width or depth of the microchannel does nothave to be constant and the microchannel may have a partially differentwidth or depth.

The detection of the passing of the sample solution through themicrochannel into a different temperature zone and the measurement offluorescence intensity for each thermal cycle are preferably performedusing the same fluorescence detector, but may be performed usingdifferent fluorescence detectors. The method for detecting the passingof the sample solution between the temperature zones is not limited tofluorescence detection and may be an optical methodology, such ascolorimetry and optical absorption, or electric methods utilizing, forexample, changes in capacitance or including electrochemical reactions.Two or more temperature zones in contact with the microchannel may be incontact from the outside of the microchannel or may be included insidethe microchannel.

In the microchannel shown in FIG. 2, two serpentine microchannels for adenaturation temperature zone and an extension/annealing temperaturezone are connected to each other via a linear intermediate-microchannel,and the passing of the sample solution and fluorescence are detected inthe internal microchannel. In FIG. 2, an intermediate-microchannel isdisposed along a straight line connecting two apertures (connections toliquid delivery mechanisms), and the passing of the test solution andfluorescence are detected in the intermediate-microchannel. Accordingly,even if the PCR chip (chip for nucleic acid amplification) is turnedupside down (turned 180 degrees) and disposed on the substrate,fluorescence can be detected using a fluorescence detector.

For example, as shown in FIG. 2, a PCR chip may be fixed to the twoheaters in such a manner that the sealing surface of each serpentinemicrochannel portion encompassed by a dotted line is attached, and,after use, the PCR chip may be removed, disposed, and replaced for theintended use. Two liquid delivery microblowers are used for eachmicrochannel of the PCR chip. Each of the microblowers is connected viaan air pressure tube to disposable tips connected to both ends of themicrochannel. The microblowers are operated alternately, thus enablingbi-directional delivery of a liquid. It is also possible tosimultaneously subject different samples to thermal cycling, forexample, to perform multiple PCR, by increasing the number of liquiddelivery microblowers in accordance with the number of microchannels.

The term “multiplex PCR” in the context of the present invention refersto PCR using a primer set comprising two or more types of forward andreverse primers in a single reaction solution. The term “primer set” inthe context of the present invention refers to a combination of one typeor two or more types of forward primers and reverse primers. In thepresent invention, a primer set comprising even only one type of reverseprimer can also be used as a primer set for multiplex PCR as long asdifferent amplification products are produced by using the reverseprimer in combination with two or more types of forward primers (asprimer pairs).

In the multiplex PCR of the present invention, fluorescence intensitiesare simultaneously measured to detect amplification of the target genescorresponding to different fluorescence wavelengths. Although detectioncan be performed using multiple fluorescence detectors, detection isfeasible even using one wavelength of light.

The present invention is described below more specifically withreference to Examples. However, the present invention is not limited tothe Examples.

Example 1: Quantification of Escherichia coli

Using a PCR chip for high-speed, real-time PCR and the device of thepresent invention, Escherichia coli (E. coli) was quantified.

Escherichia coli (DH5α strain) was cultured overnight in a Lecithinbouillon liquid medium. After an Escherichia coli suspension in aconcentration of 1×10⁴ cfu/μL was prepared based on the colony count byan agar plate medium assay, a series of 10-fold dilutions was made andused as a standard sample for quantitative identification.

The target DNA amplified in real-time PCR was a 106 bp DNA sequence ofEscherichia coli-specific uid A gene (Accession Number: NC_000913.3).Using 5′-GTG TGA TAT CTA CCC GCT TCG C-3′ (SEQ ID NO: 1) as a forwardprimer for PCR and using 5′-AGA ACG GTT TGT GGT TAA TCA GGA-3′ (SEQ IDNO: 2) as a reverse primer, the final concentration of each primer inthe PCR solution was adjusted to 300 nM. The sequence of TaqMan® probefor real-time PCR was 5′-FAM-TCG GCA TCC GOT CAG TOG CAG T-MGB-3′ (SEQID NO: 3). The final concentration of the probe in the PCR solution wasadjusted to 200 nM.

As another reagent, SpeedSTAR® HS DNA polymerase of Takara Bio, Inc. wasused in a final concentration of 0.1 U/μL. FAST Buffer I and dNTPMixture included in the product package were mixed in a concentration inaccordance with a product manual and used as a premixture for PCR. After0.5 μL of an Escherichia coli suspension in various concentrations Wasmixed with 12 μL of the premixture for PCR using a micropipette, the endof a disposable tip of the micropipette having a PCR solution absorbedtherein was inserted into an aperture at first end of the microchannelof the PCR chip and the disposable tip and the micropipette werereleased. Another empty disposable tip for the micropipette was mountedon a second end of the microchannel on the first end of which thedisposable tip for the micropipette containing the PCR solution has beenmounted. Tubes of liquid delivery microbrewers were connected to thedisposable tips. As thermal cycling conditions in high-speed, real-timePCR, a process comprising heating at 98° C. for 30 seconds for thehot-starting of DNA polymerase, further heating at 98° C. for 2 secondsand then heating at 58° C. for 4 seconds was set to be repeated for 45cycles. High-speed, real-time PCR was performed by program control ofliquid delivery microblowers.

As shown in FIG. 3, fluorescence intensity per cycle in high-speed,real-time PCR drew a sigmoid curve similar to that obtained by using aknown real-time PCR device. The fluorescence amplification rate varieddepending on the initial concentration of E. coli.

When the number of cycles that crosses the desired threshold offluorescence intensity was defined as Ct and a calibration curve wasplotted against the initial concentration of E. coli, a good linearitywas obtained, as shown in FIG. 4. Since the Ct value of the no templatecontrol (NTC), which is 0 cfu/μL, was 45 cycles or more, the resultsconfirmed that quantification is feasible even at a concentration of 100cfu/μL. The detection sensitivity was confirmed to be equivalent to thatof existing real-time PCR devices.

The high-speed, real-time PCR processing time was 6 minutes and 40seconds per 45 cycles. In contrast, the PCR processing time of even ahigh-speed thermal cycling device among existing commercially availabledevices is 45 minutes per 45 cycles. The present invention thus achieveda high-speed, real-time PCR capable of quantifying a microorganism orDNA at an extremely high speed.

Example 2: Examination of Conditions for High-speed PCR Amplification

High-speed, real-time PCR was performed by repeating three stepsconsisting of a DNA denaturation reaction, an annealing reaction, and anextension reaction in which each primer is extended from the 3′-end forreplication by a DNA polymerase in accordance with a DNA templatesequence. Among these, the DNA denaturation reaction and the annealingreaction do not depend on the length of the target DNA and are completedin a short time. However, the extension reaction requires time dependingon the length of the target DNA and enzyme activity of the DNApolymerase used. An appropriate time setting for thermal cycling is alsonecessary in high-speed, real-time PCR.

10⁴ copies of 16S ribosomal RNA gene (Accession Number KC_768803.1) ofE. coli (DH5α strain) were used as template DNA. Changing the length ofthe target DNA in the range of about 200 to 800 bp among the obtainedtemplate DNA, 45 cycles of high-speed, real-time PCR were performed.

5′-GTT TGA TCC TGG CTC A-3′ (SEQ ID NO: 4) was used as a common forwardprimer sequence and 5′-FAM-CGG GTG AGT AAT GTC TGG-TAMRA-3′ (SEQ ID NO:5) was used as a common TaqMan® probe. The following reverse primerswere used in combination therewith depending on the length of the targetDNA. The reverse primer sequence for a target DNA length of about 200 bpwas 5′-CTT TGG TCT TGC GAC G-3′ (SEQ ID NO: 6). The reverse primersequence for about 400 bp target DNA was 5′-GCA TGG CTG CAT CAG-3′ (SEQID NO: 7). The reverse primer sequence for about 600-bp target DNA was5′-CTG ACT TAA CAA ACC GC-3′ (SEQ ID NO: 8); and a reverse primersequence for about 800-bp target DNA was 5′-TAC CAG GGT ATC TAA TCC-3′(SEQ ID NO: 9). The Tm values were all set to about 50° C.

FIG. 5 is a graph plotting retention time against each target DNAlength, the retention time allowing sufficient amplification of targetDNA without exhibiting any changes in Ct values even when the annealingtime and extension reaction time are shortened. For about 100-bp shorttarget DNA, the results of the above-mentioned uid A gene were used andplotted on the same graph. FIG. 5 shows that even when the length of thetarget DNA is the same, the annealing reaction time and extensionreaction time vary depending on the activity of the DNA polymerase used.When the SpeedSTAR® HS DNA polymerase was used, the extension rate wasabout 78 bp per second. When ExTaq HS DNA polymerase was used, theextension rate was about 22 bp per second.

Theoretically, when the length of the target DNA was 0 bp, whichcorresponds to the X-intercept in FIG. 5, the annealing reaction timeexcluding the extension reaction was about 2.7 seconds, regardless ofthe kind of DNA polymerase. This result is consistent with theabove-described previous finding. Accordingly, theoretically, thehighest-speed, real-time PCR can be performed by setting the time basedon FIG. 5 in accordance with the length of the target DNA.

Example 3: Multiplex PCR High-Speed, Real-Time PCR

As an application example of high-speed, real-time PCR to a multiplexPCR method for confirming the presence or absence of multiple targetDNAs from the same sample, simultaneous detection of Neisseriagonorrhoeae, Chlamydia trachomatis, and human-leukocyte-derived β actingene was examined using a multicolor fluorescence detector that cansimultaneously detect three kinds of fluorescence.

A multicolor fluorescence detector is capable of coaxially quantifyingeach fluorescence of blue excitation, green excitation, and redexcitation. The detector can individually detect fluorescenceamplification by 3 kinds of fluorescent probes, i.e., a FAN-labeledprobe, a Texas red-labeled probe, and a Cy5-labeled probe at the samedetecting point of a microchannel on the PCR chip. Even when amulticolor fluorescence detector is used, the detector is disposed insuch a manner that 3 types of fluorescence intensity can besimultaneously detected at one detecting point on a linear microchannellocated at the center of the microchannel. When the PCR solutiondelivered from one serpentine microchannel portion by applying pressurehas passed through the detecting point, liquid delivery microblowers arestopped, so that the PCR solution can be retained in the otherserpentine microchannel for a certain period of time.

The length of the target DNA for each of Neisseria gonorrhoeae,Chlamydia trachomatis, and actin gene, and the Tm values of each of theprimers and fluorescent probes were unified so as not to make adifference in amplification efficiency.

As fluorescent probes for Neisseria gonorrhoeae, Chlamydia trachomatis,and β actin gene, Texas red-, Cy5-, and FAM-labeled TaqMan® probes wereused, and the final concentration of each probe in the PCR solution wasadjusted to 200 nM.

The final concentration of each of three kinds of forward primers andreverse primers for Neisseria gonorrhoeae, Chlamydia trachomatis, and βactin gene in the PCR solution was adjusted to 300 nM. As anotherreagent, SpeedSTAR® HS DNA Polymerase produced by Takara Bio, Inc. wasused in a final concentration of 0.2 U/μL, and FAST Buffer I and dNTPMixture included in the polymerase kit were mixed in the concentrationsspecified by the manual to form a premixture for PCR.

As template DNAs for Neisseria gonorrhoeae, Chlamydia trachomatis, and βactin, synthetic plasmids comprising each target DNA sequence wereprepared. 4 ng/μL of each plasmid was used in positive controls. Sterilewater was mixed instead of plasmid in NTC. A high-speed, real-time PCRwas thus performed. The thermal cycling conditions were such that afterheating at 96° C. for 20 seconds for hot-starting, heating at 96° C. for3 seconds and heating at 60° C. for 8 seconds were performed, and thisprocess was repeated for 45 cycles. The thermal cycling time for 45cycles under these conditions was 9 minutes and 40 seconds.

FIG. 6 shows the results of multiplex PCR for Neisseria gonorrhoeae,Chlamydia trachomatis, and β actin gene using high-speed, real-time PCR.Since the sensitivity of a multicolor fluorescence detector to threefluorescent dyes varies, the results obtained by dynamic rangecorrection are shown. In FIG. 6, the three thicker lines show changes influorescence intensities for three kinds of fluorescence when alltemplate DNA were included. Compared with fluorescent signals in NTC,which are indicated by thinner lines, clear amplification was obtainedand multiple-item simultaneous measurement from the same sample wasachieved.

In this Example, three fluorescence intensities were simultaneouslymeasured to detect the amplification of the target genes correspondingto each fluorescence wavelength. If liquid delivery microblowers arestopped immediately after the PCR solution delivered from one serpentinemicrochannel by applying pressure has passed through the detecting pointand the passing of the solution is detected, it is not necessary to useall of the fluorescence detectors, and the detection is feasible evenwith a detection signal using one wavelength of light.

Example 4: One-Step Reverse Transcription Real-Time PCR

A technique in which a reverse transcription reaction from RNA and areal-time PCR method are conveniently performed using a single reactionsolution, which is prepared by mixing a reverse transcriptase with a PCRsolution beforehand, is called a one-step reverse transcriptionreal-time PCR method. This method has been used for detecting RNAviruses, such as influenza viruses and noroviruses. In the one-stepreverse transcription real-time PCR method, two-stage steps as in ageneral RT-PCR method are combined into one to remarkably simplify theoperation. However, this method has a problem in that reversetranscriptase of the reverse transcription reaction and DNA polymeraseof the real-time PCR method interfere with each other, thus resulting inpoor PCR efficiency. However, quick shifting to the optimum temperaturesfor the activity of reverse transcriptase and DNA polymerase byhigh-speed temperature control allows the reverse transcription reactionand the real-time PCR method to be performed efficiently in order, thuseffectuating a highly efficient one-step reverse transcription real-timePCR method. Using the PCR chip for high-speed, real-time PCR and thedevice of the present invention, quantification of norovirus G1 gene andG2 gene by a one-step reverse transcription real-time PCR method wasactually examined.

As RNA comprising a target G1 gene or G2 gene sequence, a standardproduct included in a commercially available TaKaRa qPCR Norovirus(GI/GII) Typing Kit, or RNA, which is a transcription product ofsynthetic DNA, was used. A dilution series of the RNA was prepared usingan RNase-free sterile water.

The sequences disclosed in the method for detecting noroviruses providedby the Infectious Disease Surveillance Center, National Institute ofInfectious Diseases, Japan were used as the primer and probe sequences.For the norovirus G1 gene, the forward primer sequence was 5′-CGY TGGATG CGN TTY CAT GA-3′ of COG-1F (SEQ ID NO: 10); the TaqMan® probesequences were 5′-AGA TYG CGA TCY CCT GTC CA-3′ of RING1-TP (a) (SEQ IDNO: 11) and 5′-AGA TCG CGG TCT CCT GTC CA-3′ of RING1-TP (b) (SEQ ID NO:12); and the reverse primer sequence was 5′-CTT AGA CGC CAT CAT CAT TYAC-3′ (SEQ ID NO: 13). For the norovirus G2 gene, the forward primersequence was 5′-CAR GAR BCN ATG TTY AGR TGG ATG AG-3′ of COG-2F (SEQ IDNO: 14); the TaqMan® probe sequence was 5′-TGG GAG GGS GAT CGC RAT CT-3′of RING2 AL_TP (SEQ ID NO: 15); and the reverse primer sequence was5′-TCG ACG CCA TCT TCA TTC ACA-3′ (SEQ ID NO: 16) of COG-2R.

As fluorescent probes for G1 gene and G2 gene, FAM-labeled TaqMan®probes were used. The final concentration of each probe in the PCRsolution was adjusted to 200 nM.

The final concentrations of the forward primer and reverse primer forthe G1 gene or G2 gene in the PCR solution were adjusted to 300 nM. Forother reagents, PrimeScrip® Reverse Transcriptase of Takara Bio, Inc. orSuperScript® Reverse Transcriptase of Life Technologies Corporation wasused in a final concentration of 5 U/μL; an RNase inhibitor was used ina final concentration of 1 U/μL; and SpeedSTAR® HS DNA polymerase wasused in a final concentration of 0.2 U/μL. FAST Buffer I and dNTPMixture included in the product package were mixed in the concentrationsspecified by the manual, and used as a premixture for one-step reversetranscription real-time PCR.

The thermal cycling conditions were set as follows. When PrimeScrip®Reverse Transcriptase of Takara Bio, Inc. was used for the reversetranscription reaction, the reaction was performed at 42° C. for 10seconds. When SuperScript® Reverse Transcriptase of Life TechnologiesCorporation was used, the thermal cycle conditions were 55° C. for 10seconds. These reverse transcription reactions were performed in aserpentine microchannel located on a lower temperature heater of a PCRchip for high-speed, real-time PCR. After completion of the reversetranscription reaction, the temperature of the lower temperature heaterwas raised to 56° C. and the liquid was continuously delivered, wherebythe process comprising heating at 96° C. for 10 seconds for hot startingand then further heating at 96° C. for 3 seconds and at 56° C. for 8seconds was repeated for 45 cycles. The time required for 45 cycles ofone-step reverse transcription real-time PCR under these conditions was10 minutes and 20 seconds or less.

The fluorescence intensity for each cycle in the high speed, one-stepreverse transcription real-time PCR was as shown in FIG. 7. When theinitial concentrations of norovirus G1 and G2 genes are the same,similar sigmoid curves are obtained without depending on the type ofreverse transcriptase used, and the number of cycles in which thefluorescence intensity rapidly amplified and rose was the same for bothof the norovirus G1 and G2 genes.

Next, high-speed, one-step reverse transcription real-time PCR wasperformed by changing the initial concentrations of the RNA of norovirusG1 and G2 genes. As shown in FIG. 8, the number of cycles in whichfluorescence intensity rapidly amplified and rose varied depending onthe initial concentration of the norovirus G1 gene. The number of cyclesin which rapid amplification and rise of the fluorescence intensity areobserved is in the order of highest to lowest initial concentration ofRNA. Accordingly, the initial concentration of RNA can be generallyquantified through Ct value, which is the number of cycles in whichfluorescence intensity rapidly amplifies and rises.

However, the amplification curve for the norovirus G1 gene shown in FIG.8 confirms that the baseline is an upward-sloping curve in such a mannerthat the fluorescence intensity amplifies gently in accordance withthermal cycling, and then the fluorescence intensity rapidly increasesfrom a certain number of cycles. Accordingly, it is difficult toestimate an accurate Ct value by using a usual method in which a certainlevel of fluorescence intensity is defined as the threshold and thenumber of cycles that provides a fluorescence intensity higher than thethreshold is defined as the Ct value.

Accordingly, in order to detect the Ct value during the one-step reversetranscription real-time PCR even when the baseline is not a constantvalue, i.e., the baseline increases proportionally, the Ct value wasdeduced from a matrix of fluorescence intensity (two-dimensional arrayof the amplification curve) determined for each number of thermalcycles.

To detect the slope that rapidly rises relative to the slope below theCt value in a two-dimensional array of the amplification curve, thefollowing may be performed. When there is a great variation influorescence intensity, the running average may be obtained, ifnecessary. While doing so, a first forward differentiation is performedfor each thermal cycle. Of the new two-dimensional array of the obtainedslope, the root mean square (which may alternatively be the weightedaverage) of the slope of the initial stage (for example, 5 to 15 cycles,or 5 to 15 cycles immediately before each cycle) and the slope after theinitial stage were compared. When a significant (for example, 5-fold ormore, which may alternatively be 2-fold or more) increase in the numberof cycles was observed, it was deduced as the number of cycles Ct inwhich the fluorescence intensity rapidly amplifies and rises.

FIG. 9 shows calibration curves prepared from the obtained Ct valuesagainst initial concentrations of norovirus G1 and G2 genes. Goodlinearity is obtained relative to each RNA concentration of thenorovirus G1 and G2 genes. The Ct value can be promptly determined evenduring one-step reverse transcription real-time PCR, when fluorescenceintensity rises rapidly. The initial RNA concentration can be calculatedfrom the Ct value.

A feature of the present invention is a system in which the entire PCRsolution passes through a microchannel in such a manner that thesolution passes a fluorescence detecting point for each thermal cycle.Accordingly, even if a fluorescent dye generated by real-time PCR isnon-uniformly dispersed as a fluorescent dye concentration in a PCRsolution due to a lack of time to uniformly disperse the fluorescent dyein the PCR solution, which results from faster thermal cycling, all thefluorescent dyes is detected by a fluorescence detector and integratedto thereby accurately quantify the fluorescence amount for each cycle.

Accordingly, as shown in FIG. 9, in the calibration range, error barsfor Ct values in the measurement of RNA concentration are very small andexact quantification with excellent reproducibility is confirmed to befeasible.

INDUSTRIAL APPLICABILITY

The device according to the present invention is transportable andallows high-speed, real-time PCR to be performed at low cost in aclinical setting or on the spot where an infectious disease occurs. Morespecifically, the spread of infection can be prevented by quickconfirmation of therapeutic effects and early detection of infectiousdiseases in livestock and poultry.

1. A reciprocal-flow-type nucleic acid amplification device comprising:heaters capable of forming a denaturation temperature zone and anextension/annealing temperature zone; a fluorescence detector capable ofdetecting movement of a sample solution between the two temperaturezones; a pair of liquid delivery mechanisms that allow the samplesolution to move between the two temperature zones and that areconfigured to be open to atmospheric pressure when liquid deliverystops; a substrate on which a chip for nucleic acid amplification can beplaced; and a control mechanism that controls driving of each liquiddelivery mechanism by receiving an electrical signal relating tomovement of the sample solution from the fluorescence detector, thedevice being capable of performing real-time PCR by measuringfluorescence intensity for each thermal cycle.
 2. A chip for nucleicacid amplification comprising at least one microchannel, themicrochannel comprising: curved-microchannels each for the denaturationtemperature zone and the extension/annealing temperature zone of thenucleic acid amplification device according to claim 1; a linearintermediate-microchannel connecting the curved-microchannels; andconnections at both ends of the microchannel, the connections beingconnectable to the liquid delivery mechanisms of the nucleic acidamplification device according to claim
 1. 3. The nucleic acidamplification device according to claim 1, wherein the liquid deliverymechanisms are microblowers or fans.
 4. A nucleic acid amplificationmethod comprising the following steps: step 1: placing the chip fornucleic acid amplification according to claim 2 on the substrate in sucha manner that the denaturation temperature zone includes onecurved-microchannel and the extension/annealing temperature zoneincludes another curved-microchannel; step 2: introducing a samplesolution into the microchannel; step 3: connecting a pair of liquiddelivery mechanisms to liquid delivery mechanism connections at bothends of the microchannel; and step 4: reciprocating the sample solutionbetween the two curved-microchannels of the microchannel by the liquiddelivery mechanisms to perform thermal cycling, and simultaneouslymeasuring the fluorescence intensity of the sample solution andconfirming the passing of the sample solution for each thermal cycleusing at least one fluorescence detector in theintermediate-microchannel to perform real-time PCR.
 5. The nucleic acidamplification method according to claim 4, wherein the measurement offluorescence intensity is performed by simultaneously measuring two ormore fluorescent wavelengths to simultaneously measuring real-time PCRof multiple genes in one microchannel.
 6. The nucleic acid amplificationmethod according to claim 4, wherein the measurement of fluorescenceintensity is performed using a calibration curve obtained from thenumber of cycles Ct derived from a fluorescence intensity matrix perthermal cycle (a two-dimensional array of an amplification curve). 7.The nucleic acid amplification method according to claim 4, wherein thenucleic acid amplification method is selected from the group consistingof polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR),one-step RT-PCR, multiplex PCR, multiplex RT-PCR, real-time PCR, andreal-time RT-PCR.
 8. The nucleic acid amplification method according toclaim 4, comprising performing interruption analysis, which is madepossible by forming two or more microchannels on a flat substrate insuch a manner that the operation of liquid delivery through each of themicrochannels can be independently controlled.
 9. The nucleic acidamplification method according to claim 4, the method comprising:connecting an end of a filtered pipette tip of a micropipette to one ofthe connections so as to introduce a sample solution into themicrochannel; removing the micropipette with the pipette tip beingconnected to the connection, and then connecting the pipette tip to oneof the liquid delivery mechanisms.
 10. The nucleic acid amplificationmethod according to claim 4, wherein the volume of the sample solutionintroduced into the microchannel is in the range of 5 μL to 50 μL. 11.The chip for nucleic acid amplification according to claim 2, the chipbeing for use in the nucleic acid amplification method comprising thefollowing steps: step 1: placing the chip for nucleic acid amplificationaccording to claim 2 on the substrate in such a manner that thedenaturation temperature zone includes one curved-microchannel and theextension/annealing temperature zone includes anothercurved-microchannel; step 2: introducing a sample solution into themicrochannel; step 3: connecting a pair of liquid delivery mechanisms toliquid delivery mechanism connections at both ends of the microchannel;and step 4: reciprocating the sample solution between the twocurved-microchannels of the microchannel by the liquid deliverymechanisms to perform thermal cycling, and simultaneously measuring thefluorescence intensity of the sample solution and confirming the passingof the sample solution for each thermal cycle using at least onefluorescence detector in the intermediate-microchannel to performreal-time PCR.
 12. The nucleic acid amplification method according toclaim 5, wherein the measurement of fluorescence intensity is performedusing a calibration curve obtained from the number of cycles Ct derivedfrom a fluorescence intensity matrix per thermal cycle (atwo-dimensional array of an amplification curve).
 13. The nucleic acidamplification method according to claim 12, wherein the nucleic acidamplification method is selected from the group consisting of polymerasechain reaction (PCR), reverse transcription PCR (RT-PCR), one-stepRT-PCR, multiplex PCR, multiplex RT-PCR, real-time PCR, and real-timeRT-PCR.
 14. The nucleic acid amplification method according to claim 13,comprising performing interruption analysis, which is made possible byforming two or more microchannels on a flat substrate in such a mannerthat the operation of liquid delivery through each of the microchannelscan be independently controlled.
 15. The nucleic acid amplificationmethod according to claim 14, the method comprising: connecting an endof a filtered pipette tip of a micropipette to one of the connections soas to introduce a sample solution into the microchannel; removing themicropipette with the pipette tip being connected to the connection, andthen connecting the pipette tip to one of the liquid deliverymechanisms.
 16. The nucleic acid amplification method according to claim15, wherein the volume of the sample solution introduced into themicrochannel is in the range of 5 μL to 50 μL.